In the assay of body fluids, especially blood serum, for triglyceride concentration, the initial step requires hydrolysis of triglycerides, for example, hydrolysis of triglycerides to glycerol.
Conventional procedures for triglyceride hydrolysis use a strong base (KOH, NaOH, etc.), or for reasons of simplicity and selectivity, a hydrolase enzyme (i.e., a lipase). Handling of caustic materials may be inconvenient or undesirable and, as discussed in relation to prior publications below, enzymatic techniques can be useful for the hydrolysis of "free" triglycerides, i.e., those not bound to protein, but are either ineffective or very slow when used to treat protein-bound triglycerides. The binding of the triglyceride to protein apparently inhibits the action of the hydrolase and thus requires some means for breaking the protein-triglyceride complex before the hydrolase can act on the triglyceride.
U.S. Pat. No. 3,703,591 to Bucolo et al describes the use of a combination of a lipase and a protease to achieve serum (i.e., protein-bound) triglyceride hydrolysis. No suggestion is made to use a surfactant either in combination with or as a substitute for the protease.
U.S. Pat. No. 3,759,793 to Stork et al describes the hydrolysis of serum triglycerides using a lipase from Rhizopus arrhizus which is apparently identical to that suggested by Bucolo et al, however, with no requirement for a protease. The reasons for this apparent anomaly are not clear, however, it is noted in British Pat. No. 1,395,126 of the same assignee that the Stork et al hydrolysis technique is very slow. This British Patent describes an improved method for hydrolyzing triglycerides with the aforementioned Rhizopus arrhizus lipase comprising contacting the triglyceride with the lipase in a buffer and in the presence of carboxylesterase and an alkali metal or alkaline earth metal alkyl sulfate, the alkyl radical of which contains 10 to 15 carbon atoms. In support of the patentability of this technique this patent cites the slowness of earlier enzymatic techniques, specifically those of Stork et al. The preferred alkyl sulfate is sodium dodecyl sulfate. There is no suggestion that the use of surfactant alone in the absence of carboxylesterase stimulates lipase activity and in fact, as will be shown in the examples below, the use of at least some of the stimulating surfactants described herein actually inhibit the activity of lipase from Rhizopus arrhizus.
Helenius, Ari and Simons, Kai, Biochemistry, Vol. 10, No. 13 (1971) describe a method for removing all major lipids from human plasma low-density lipoprotein comprising treatment of the human plasma with high concentrations of natural and synthetic surfactants. Lipid removal is applied for purposes of characterizing the lipid free protein moiety of human plasma low-density lipoprotein. There is no suggestion in this publication that the combination of a surfactant and a lipase would yield a useful analytical tool which would simplify the assay of serum for triglyceride content by providing a fast and accurate hydrolysis method and stable assay compositions.
U.S. Pat. No. 3,689,364 issued Sept. 5, 1972 describes an assay for lipase contained in body fluids such as blood serum using a "free" triglyceride emulsion as substrate for the lipase. It is suggested that the bile salts which stabilize the substrate emulsion of "free" triglyceride (i.e., triglycerides not bound to protein) also concurrently exert an "activating effect" on the lipase under assay when it is a pancreatic lipase. The activating effect apparently results in an increase in the hydrolytic activity of the lipase on the free triglycerides of the substrate emulsion. There is no teaching or suggestion in this patent that such bile salts exert any effect on lipase preparations when contacted with lipids bound to proteins as are found in blood serum.
U.S. Pat. No. 3,898,130 to Komatsu issued Aug. 5, 1975 describes a method for hydrolyzing triglycerides comprising contacting triglyceride with a composition comprising a mixture of a microbial lipase, particularly Candida lipase, a pancreatic lipase and a bile salt. Both the microbial and the pancreatic lipase enzymes are critical components of the hydrolysis composition.
German Offenlegungsschrift No. 2,522,432 published Dec. 4, 1975 describes an enzymatic method for hydrolyzing cholesterol esters using a cholesterol esterase from Pseudomonas fluorescens.
U.S. Pat. No. 3,925,164 issued Dec. 9, 1975 describes a method for hydrolyzing cholesterol esters using a cholesterol esterase from Candida rugosa, Rhizopus and Aspergillus. It is suggested that the addition of a surfactant increases the activity of the esterase.